1446 Vol. 42, No. 9Biol. Pharm. Bull. 42, 1446–1449 (2019)

© 2019 The Pharmaceutical Society of Japan

Communication to the Editor

Flavonoids with Two OH Groups in the B-Ring Promote Pigmented Hair RegenerationNobuhiko Taguchi,a,b Minoru Yuriguchi,a Takuya Ando,a Ryosuke Kitai,a Hitomi Aoki,a and Takahiro Kunisada*,a

a Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine; Gifu 501–1194, Japan: and b General Research & Development Institute, Hoyu Co., Ltd.; Aichi 480–1136, Japan.Received April 2, 2019; accepted May 28, 2019

During the process of skin regeneration following a skin injury, de novo hair follicle regeneration is initiated after wounding; however, these regenerated hairs are mostly un-pigmented. The activation of epidermal melanocyte stem cells and their differentiation into regenerating hair follicles have been shown to be necessary for the pigmented hair regenera-tion after wounding. To determine the role of flavonoids in the regeneration of pigmented hairs, we applied the candidate fla-vonoids to the regenerating hair follicles after wounding and identified the flavonoid species that maximally induced pig-mented hair regeneration. Flavonoids with two OH groups in the B-ring, such as sterubin, luteolin, and hydroxygenkwanin, showed promising effects in regenerating black pigmented hairs, while those with one OH group in the B-ring showed no significant change. Thus, flavonoids with two OH groups in their B-ring could be studied further as potential wound healing agents with the ability to regenerate pigmented hair.

Key words  flavonoid;  wound  healing;  pigmentation;  hair  re-generation

INTRODUCTION

During wound  healing  in  the  skin,  hair  follicles  are  regen-erated  under  severe wounding  conditions.1) In adult mice, re-generated hairs are mostly unpigmented because follicular me-lanocytes are not activated during the regeneration process.1) Recently, melanocyte stem cells have been reported to respond to  wounding  and  migrate  to  the  nearby  epidermis  from  the existing hair follicles residing very close to the regenerating skin. These skin regeneration-associated melanocytes have the potential to differentiate into follicular melanocytes of regenerating hair follicles to form pigmented hairs.2,3) Wnt signaling,  first  produced  in  the  follicular  keratinocyte  stem cells,  is  known  to  stimulate  both  hair  follicular  keratinocytes and follicular melanocyte stem cells to induce coordinated pigmented hair  follicle  regeneration after wounding.3) Further-more,  efficient  regeneration  of  pigmented  hairs  was  observed when  mice  were  wounded  at  5  weeks  after  birth.  During this  process,  the  follicles  were  in  the  anagen  phase,  and  the expression  of Wnt7a,  a  known  inducer  of  wound  healing  as-sociated with hair follicle regeneration, was highest in the epi-dermis.4) Pigmented hair regeneration markedly reduced after wounding  during  the  telogen  phase  at  7  weeks  after  birth. However, when  drugs  activating Wnt  signaling,  such  as LiCl, were applied to the wounded skin during this period, a higher proportion of pigmented hairs in the regenerated hair follicles was observed.4)

Flavonoids are plant-derived polyphenolic compounds claimed to help alleviate a variety of conditions owing to their antioxidant,  anti-inflammatory  effects,  wound  healing,  and melanogenesis effects. Luteolin treatment has been reported to promote wound healing possibly via its epithelization promot-ing and free-radical scavenging activities.5) In our previous study,  sterubin  was  found  to  promote  melanin  production through  the  Wnt  signaling  pathway  in  the  human  melanoma cell line HMVII and prevent the progression of hair graying.6) Luteolin,  diosmetin,  and  hesperetin  are  known  to  promote 

* To whom correspondence should be addressed.  e-mail: tkunisad@gifu-u.ac.jp

Table 1. Flavonoids and Their Structures

The skeleton structure of flavanone and flavone with  the rings named. The chemical structure diagrams were drawn manually using ISIS/Draw (MDL Information Systems, Inc., CA, U.S.A.).

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Fig. 1. Regeneration of Pigmented Hairs after Wounding(A) Representative photos of regenerated dorsal skin area at 80 d after wounding. Scale bar = 1 mm. (B) The percentage of regenerated hairs and their pigmentation. The

flavonoids (0.1%w/v, 200 µL/d) were applied to the wounded area for one week. Hair regeneration and hair color were assessed under a microscope at 80 d after resection. Hairs only on re-epithelialized areas containing at least 10 regenerated hairs were counted, and only areas with more than one-third of regenerated hairs with pigmentation were designated as areas containing pigmented regenerated hairs. The number of mice used for the experiments is shown. The results were analyzed using chi-square test (hair regeneration: * p < 0.05, ** p < 0.01, pigmented hair regeneration: †† p < 0.01).

1448 Vol. 42, No. 9 (2019)Biol. Pharm. Bull.

melanin production in cultured cells; therefore, their effects on the darkening of hair are being considered.7–9)

In this study, flavonoids with two OH groups in the B-ring, such as sterubin,  luteolin, hydroxygenkwanin (HGK), and eri-odictyol, and one OH group in the B-ring, such as hesperetin, homoeriodictyol, and diosmetin, that possibly have the poten-tial  to  induce  regeneration  of  pigmented  hairs  during  wound healing were  evaluated,  and  their  effects were  compared with each  other. We  hypothesized  that  flavonoids  applied  onto  the wounded  skin  of  mice  can  induce  appropriate  signals  to  ac-tivate follicular keratinocytes and melanocytes necessary for regeneration  of  the  pigmented  hairs  and/or  to  prevent  inflam-mation during the regeneration process; therefore, a higher proportion of pigmented hairs could be expected in the treated regenerated hair follicles.

MATERIALS AND METHODS

Dorsal  skin  samples  (2.25 cm2)  were  excised  from  7-week-old C57BL/6 mice  after  trimming  the  surrounding  hairs with electric  clippers.  All  animal  experiments  were  approved  by the  Animal  Research  Committee  of  the  Graduate  School  of Medicine, Gifu University. The flavonoids (0.1%w/v) shown in Table 1 were dissolved  in 50% ethanol, and 200 µL of  the fla-vonoid solution was applied daily to the wounded area for one week. Sterubin used was synthethyzed in  the  laboratory,6) and the other flavonoids: luteolin (LKT Laboratories, MN, U.S.A.), hydroxygenkwanin  (Phytolab,  Vestenbergsgreuth,  Germany), eriodictyol  (Extrasynthese,  Lyon,  France),  hesperetin  (Alexis Corporation, Nottingham, U.K.), homoeriodictyol (Extrasyn-these) and diosmetin (LKT Laboratories) were all commercial-ly  available  products.  Hair  regeneration  and  hair  color  were assessed using a microscope (Moritex Corporation, Saitama, Japan) at 80 d after resection. Hairs only on re-epithelialized areas  containing  at  least  10  regenerated  hairs  were  counted, and  only  areas with more  than  one-third  of  pigmented  regen-erated  hairs  were  designated  as  areas  containing  pigmented regenerated hairs.4)

RESULTS AND DISCUSSION

To evaluate the pigmented hair regeneration during the wound  healing  process  of  the  mouse  treated  with  flavonoids, we  shaved  the  hair  around  the  reepithelialized  areas  and  then photographed the regenerated hair. On day 80, the rate of hair regeneration including black pigmented and unpigmented hairs  was  as  follows:  no  drug  (20.5%),  50%  ethanol  (22.7%), sterubin  (70.6%),  luteolin  (58.8%),  HGK  (42.9%),  eriodictyol (42.1%),  hesperetin  (11.1%)  homoeriodictyol  (9.5%),  and  dios-metin (5.3%). Sterubin and luteolin significantly activated hair regeneration,  but  homoeriodictyol  and  diosmetin  significantly inhibited  hair  regeneration  in  comparison  with  the  other  fla-vonoids and controls (Figs. 1A, 1B). Furthermore, the ratio of pigmented  hair  regeneration  was  as  follows:  no  drug  (0.0%), 50%  ethanol  (2.3%),  sterubin  (47.1%),  luteolin  (52.9%),  HGK (42.9%),  eriodictyol  (21.1%),  hesperetin  (5.6%)  homoeriodic-tyol (4.8%), and diosmetin (5.3%). Sterubin, luteolin, and HGK activated melanocyte stem cells to successfully regenerate pigmented  hairs  in  comparison with  the  other  flavonoids  and controls (Figs. 1A, B). Horibe et al. reported that the one OH group-containing diosmetin induced melanogenesis to a

greater  extent  than  the  two  OH  groups-containing  luteolin in B16F10 cells by upregulating tyrosinase through cAMP response element binding protein (CREB).7) In our study, how-ever,  the  one OH group-containing  flavonoids  did  not  signifi-cantly induce overall hair follicle regeneration. Regeneration of  hair  follicles  after wounding  requires  the  concerted  activa-tion of melanocyte stem cells, including their differentiation, and the stimulation of melanin synthesis.10) Therefore, the one OH  group-containing  flavonoids  that  had  the  least  effect  on hair regeneration are not considered suitable.Flavonoids  with  two  OH  groups  in  the  B-ring  have  been 

reported  to have a greater antioxidant activity  than  those with one OH group in the B-ring.11)  Sterubin  (two OH groups)  has been  shown  to  have  the  highest  antioxidant  activity  among flavonoids  isolated  from  the  resinous exudate of Heliotropium sinuatum and Eriodictyon angustifolium.6,12,13) Sterubin and eriodictyol  (two  OH  groups)  have  greater  neuroprotective effects than homoeriodictyol (one OH group). In addition, sterubin  showed  three  times  more  anti-inflammatory  activity than eriodictyol, and O-methylation on the A-ring increases the overall lipophilicity, bioavailability, and metabolic stability of sterubin.14) The two OH groups in the B-ring also inhibited the phosphorylation of protein kinase Cδ (PKCδ) than no or one OH group in the B-ring.11) The  two OH groups  in  the B-ring could bind with C1B domain of PKCδ phorbol ester bind-ing site better than the others and thus expected to effectively reduce PKCδ activity. Because proliferation of melanocytes is inversely  related  with  PKCδ activity,15)  two  OH  groups-con-taining flavonoids are expected to increase hair pigmentation.Pigmented  hair  regeneration  after  wounding  has  recently 

been reported  in a geriatric patient with a  large wound on  the scalp.16) To date, there have been no reports on pigmented hair regeneration in human skin after wounding.In conclusion, flavonoids with two OH groups in the B-ring 

effectively  regenerate  pigmented  hairs  after  skin  wounding, whereas  those with  one OH  group  in  the  B-ring  do  not.  Our finding may guide  further  exploration of novel  agents  that  in-duce pigmented hair regeneration.

Acknowledgment This study received funding from Hoyu Co., Ltd.

Conflict of Interest Nobuhiko Taguchi is an employee of Hoyu Co., Ltd. The other  authors declare no  conflict  of  inter-est.

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